Deciphering the rules governing synaptic communication among neurons is believed to provide the key to understanding how the brain works. Accumulating evidence supports, however, the novel view that the brain should not be regarded simply as a circuit of actively interacting neurons but rather as a network of neurons and astrocytes that intesively cooperate to perform computational feats. Astrocytes respond to the synaptic release of neurotransmitters with intracellular Ca2+ elevations mediated mainly by G-protein coupled receptors, and with the release of neuroactive molecules, collectively termed gliotransmitters, that contribute to the modulation of synaptic transmission and plasticity. The role of neuron-astrocyte interactions has been, however, intensively studied in relation to glutamatergic synaptic transmission, but little has been revealed about the role of astrocytes in GABAergic inhibitory transmission. Indeed, whether the different GABAergic interneurons specifically signal to astrocytes and what impact on the activity of local neuronal circuits this signalling pathway may are fundamental questions that have been poorly addressed. In my thesis I started to investigate the signaling between different interneurons and astrocytes, focusing on Parvalbumin (PV)- and Somatostatin (SOM)-expressing interneuron subpopulations that compose up to 70 % of the total number of GABAergic interneurons in the brain. To this aim, I developed a complex approach that combines single and two-photon laser-scanning microscopy for Ca2+ imaging, both in somatosensory cortex (SSCx) slices and in vivo, patch-clamp recording and optogenetic techniques. I found that in somatosensory and temporal cortex slices loaded with the Ca2+ indicator Fluo-4 AM and the astrocytic marker SR101, about 60 % of layer V astrocytes showed large amplitude somatic Ca2+ increases in response to GABA or baclofen (Bac, a GABAB receptor agonist) in both young and adult mice. These Ca2+ responses were abolished in mice lacking the inositol-1,4,5-trisphosphate (IP3) receptor type 2 in astrocytes, while blocking Gi/o proteins with pertussis toxin prevented Bac-mediated Ca2+ transients. These results reveal an involvement of the Gq/IP3 cascade and suggest possible Gi/o-Gq protein interactions in the astrocyte response to GABA signals.
In a mouse model in which astrocytes selectively express the genetically encoded Ca2+ indicator GCaMP3, I also found that local GABA or Bac applications induced long-lasting Ca2+ oscillations at fine processes that occasionally spread to the entire astrocytic soma and other processes. I then validated the responsiveness of astrocytes to GABAergic signals in in vivo experiments from P30-60 anesthetized GCaMP3 animals where Bac locally applied to primary SSCx layers I/II evoked Ca2+ elevations in 45.46 % ± 8.07 % of the total astrocytes observed.
Optogenetic stimulation of ChR2-expressing PV or SOM interneurons also evoked astrocytes Ca2+ events (the average of Ca2+ peaks per minute significantly increase from 0.15 ± 0.06 to 0.30 ± 0.05 for PV interneurons stimulation and from 0.19 ± 0.04 to 1.16 ± 0.13 for SOM interneurons stimulation). Current pulse depolarization of a single PV or SOM interneuron increased Ca2+ peaks in nearby astrocytes from 0.41 ± 0.04 to 0.65 ± 0.08 (p<0.05) and event frequency per minute from 0.10 ± 0.31 to 1.09 ± 0.16 (p<0.01), respectively.
Patch-clamp recordings in the presence of TTX showed that GABAB activation triggered glutamate release in astrocytes and NMDAR-mediated slow inward currents (SICs) in nearby neurons. The frequency of SICs was strongly increased both in PV interneurons (from 0.15 ± 0.06 to 0.46 ± 0.04 event/min) and pyramidal neurons (from 0.30 ± 0.07 to 0.79 ± 0.17 event/min). The increase in SICs frequency lasted for about two minutes on average, outlasting the time of GABA agonist applications. As revealed in experiments from IP3R2 KO mice, GABA-induced SICs were also dependent on IP3R mediated intracellular Ca2+ transients in astrocytes. These data suggest that astrocytes activated by GABAergic interneurons convert a transient inhibition into a delayed excitation in local circuits.
I conclude that cortical astrocytes can be activated by two of the major GABAergic interneuron classes in the brain (PV and SOM). The consequent gliotransmitter release provides a new form of homeostatic control of local network excitability
